The major goal of this project is to elucidate the mechanisms by which synapses are formed. Synapses are the cellular basis of neural circuitry and therefore are fundamental to nervous system function. A cardinal feature of the chemical synapse is the presence of a postsynaptic apparatus containing high concentrations of neurotransmitter receptors closely associated with numerous extracellular, transmembrane, and cytoplasmic scaffolding and signaling components. Perhaps best studied in this respect is the aggregation of acetylcholine receptors (AChRs) at postsynaptic sites of the vertebrate skeletal neuromuscular junction (NMJ) along the central band of the muscle. In the last several decades many studies have led to a neurocentric model in which the specialization of this postsynaptic apparatus is orchestrated by three nerve-derived signals that cluster pre-existing AChR molecules, selectively induce AChR gene expression at synaptic sites and suppress AChR gene expression at extra-synaptic sites. Based on this model, agrin, neuregulin (NRG) and acetylcholine (ACh) were identified as candidate molecules to mediate these three cellular activities, respectively. We have used mouse genetics to determine physiological roles of these three pathways. The results revealed that the muscle intrinsically signals for the initiation of postsynaptic differentiation at the central region of the muscle while the nerve and/or accompanying Schwann cells provide both positive and negative signals that promote differentiation and maintain central location and stability of synapses. In the present proposal, we will focus on understanding the role of ACh and agrin signaling pathways and their interplay in both pre-and post-synaptic development. There are three aims. Aim 1 is to determine the role of target-derived trophic factors in NMJ development in the ChAT mutant mice. Aim 2 is to determine the role of agrin in postsynaptic differentiation in the ChAT mutants. Aim 3 is to determine the role of protein phosphorylation regulated by the interplay of ACh and agrin signaling pathways in NMJ development. The results from this study will advance our knowledge on neuromuscular synapse formation and maintenance and will provide crucial information in designing treatments for neuromuscular diseases and spinal cord injury.